Journal: APL Bioengineering

Article Title: Microarrayed human bone marrow organoids for modeling blood stem cell dynamics

doi: 10.1063/5.0092860

Figure Lengend Snippet: Dynamics and mechanism of CD34 + HSPC homing. (a) Schematic illustration of the homing dynamics of HSPCs (red) into BMOs (magenta/beige/gray). (b) Representative confocal images of CFSE-labeled HSPCs (CFSE, green) and the network (CD31, magenta) within BMOs (DAPI, blue) at 4, 8, 12, 16, 20, 24, 48, 72, and 96 h after seeding of HSPCs. The maximum intensity projections of z-stack acquisitions are shown. Scale bar, 100 μ m. (c) Quantitative analysis of the number of CFSE + cells per BMO over time. Results of two independent experiments. Symbols indicate mean values, and the error bars show the standard deviations. (d) Representative confocal images of the inhibition of homing of HSPCs into BMOs 24 h after seeding (left panel). The purified HSPCs (CFSE, green) are treated with either AMD3100 or SB290157 before seeding on BMOs. In the third condition, BMOs are treated with anti-PTN before seeding of the HSPCs to negatively influence the migration behavior of the HSPCs. The network is marked with CD31 (magenta) and the nuclei with DAPI (blue). The maximum intensity projections of z-stack acquisitions are shown. Scale bar, 100 μ m. The number of homed CFSE + cells is quantified per BMO per treatment (right panel). Violin plots represent the distributions, red horizontal lines indicate the medians, and black dotted lines the quartiles. Results of two independent experiments. Statistical analysis by ordinary one-way ANOVA and multiple comparisons.

Article Snippet: CD34 + leukemic blasts were cultured in StemSpan TM SFEM II serum-free medium (STEMCELL Technologies, 09605) supplemented with the StemSpan CD34 + Expansion supplement (STEMCELL Technologies, 02691) and 1% P/S.

Techniques: Labeling, Inhibition, Purification, Migration

Journal: APL Bioengineering

Article Title: Microarrayed human bone marrow organoids for modeling blood stem cell dynamics

doi: 10.1063/5.0092860

Figure Lengend Snippet: Bone marrow organoids as a niche for leukemic cells. (a) Representative confocal images of leukemic blast cells (CFSE, green) and the network (CD31, magenta) within BMOs (DAPI, blue) at 4, 8, 12, 16, 20, 24, 48, 72, and 96 h after their addition. The maximum intensity projections of z-stack acquisitions are shown. Scale bar, 100 μ m. (b) Quantitative analysis of the number of CFSE + cells per BMO over time. Results of two independent experiments. Symbols indicate mean values, and the error bars show the standard deviations. (c) Quantification of the CFSE intensity of individual homed leukemic stem cells (LSCs) over 96 h. Representative data from one out of three independent experiments. Violin plots represent the smoothened distributions with individually analyzed CFSE + cells represented as gray dots. Dotted horizontal lines indicate the medians.

Article Snippet: CD34 + leukemic blasts were cultured in StemSpan TM SFEM II serum-free medium (STEMCELL Technologies, 09605) supplemented with the StemSpan CD34 + Expansion supplement (STEMCELL Technologies, 02691) and 1% P/S.

Techniques:

Journal: Science Advances

Article Title: Regulation of eIF4E guides a unique translational program to control erythroid maturation

doi: 10.1126/sciadv.add3942

Figure Lengend Snippet: ( A ) HUDEP-2 cells transduced with retrovirus encoding eIF4E [pMSCV-eIF4E-IRES-GFP (green fluorescent protein)] or The RNAi Consortium (TRC) control (pMSCV-TRC-IRES-GFP) followed by GFP sorting, precursor expansion, and differentiation. ( B ) Representative flow cytometry plots comparing HUDEP-WT and HUDEP-eIF4E cells 4 days after induction of maturation. ( C to E ) Quantitation of flow cytometry–defined differences in maturation between WT and eIF4E HUDEP-2 cells by (C) CD71 and FSC, (D) CD34 and CD105, and (E) cKit. * P < 0.05, N = 3. ( F ) Representative Wright-Giemsa–stained cytospins of day 0 and +4 HUDEP-WT and HUDEP-eIF4E cells displaying relative homogeneity in day 0 of both conditions (top) and day +4 (bottom) showing marked anisocytosis in HUDEP-WT cells compared to HUDEP-eIF4E cells. Green arrowheads denote erythroblasts showing nuclear condensation and size restriction. Magnification, ×40. Black line indicates size marker of 100 μm. ( G ) Representative day +4 cell pellet of WT versus eIF4E HUDEP-2 cells showing impaired hemoglobinization evidenced by the pale color of the pellet. Statistical analysis between populations using paired t test compared to HUDEP-WT. N.S., not significant.

Article Snippet: Thawed cells were plated on retronectin-coated wells in CD34 + expansion media [StemSpan SFEM II, hSCF (100 ng/ml; Peprotech), Flt3 ligand (100 ng/ml; Peprotech), Thrombopoietin (TPO) (50 ng/ml; Peprotech), low-density lipoprotein (10 μg/ml; STEMCELL Technologies), UM171 (35 nM; Benchchem), and penicillin/streptomycin (Thermo Fisher Scientific)] for 48 hours of expansion before retroviral transduction with pMSCV-IRES-GFP or pMSCV-eIF4E-IRES-GFP as described above.

Techniques: Transduction, Flow Cytometry, Quantitation Assay, Staining, Marker

Journal: Science Advances

Article Title: Regulation of eIF4E guides a unique translational program to control erythroid maturation

doi: 10.1126/sciadv.add3942

Figure Lengend Snippet: ( A ) HUDEP-2 WT and eIF4E cells nucleofected with RNP containing Cas9 with crRNA:tracrRNA duplex specific to either noncoding negative control, PTPN6, or Igf2bp1. Single-cell clones were generated and expanded before induction of maturation. ( B ) Representative flow cytometry plots of CD71 versus FSC and CD34 versus CD105 expression demonstrating the reversal of erythroid maturation in HUDEP-eIF4E (negative CRISPR control) cells with simultaneous knockdown of PTPN6 (4E PTPN6 1 or 4E PTPN6 2) or Igf2bp1 (4E Igf2bp1 1 or Igf2bp1 2) in comparison to HUDEP-WT (negative CRISPR control). ( C ) Partial rescue of CD71 + erythroid maturation phases dependent on PTPN6 knockdown. * P < 0.05, N = 6. ( D ) Partial rescue of CD71 + erythroid maturation phases dependent on Igf2bp1 knockdown. * P < 0.05, N = 6. ( E ) Partial rescue of CD34 + , CD105 + and CD34 − , CD105 + populations with knockdown of PTPN6 or Igf2bp1. * P < 0.05, N = 6. Statistical analysis between populations calculated using paired t test compared to HUDEP-4E negative control (4E Ng Cnt).

Article Snippet: Thawed cells were plated on retronectin-coated wells in CD34 + expansion media [StemSpan SFEM II, hSCF (100 ng/ml; Peprotech), Flt3 ligand (100 ng/ml; Peprotech), Thrombopoietin (TPO) (50 ng/ml; Peprotech), low-density lipoprotein (10 μg/ml; STEMCELL Technologies), UM171 (35 nM; Benchchem), and penicillin/streptomycin (Thermo Fisher Scientific)] for 48 hours of expansion before retroviral transduction with pMSCV-IRES-GFP or pMSCV-eIF4E-IRES-GFP as described above.

Techniques: Negative Control, Clone Assay, Generated, Flow Cytometry, Expressing, CRISPR

Journal: Nature metabolism

Article Title: DNA methylation mediates HbA1c-associated complications development in Type 1 diabetes

doi: 10.1038/s42255-020-0231-8

Figure Lengend Snippet: a , Genomic location of the depicted region at/near TXNIP . b , Manhattan plots of association between mean-DCCT HbA1c and DNAme at the covered CpGs. Multiple linear regression models adjusting for covariates were applied to each CpG across all the samples (two-sided tests based on t-statistic, n=499). X-axis represents CpG location and Y-axis represents –log(p). Two HbA1c-assoc regions are highlighted with pink background. c , Heatmap showing the 15 chromatin states (top) and 18 states (bottom). Colors are shown in the legends ( panel j ). d , Ribbon plots representing the genomic interactions identified by IHEC PCHi-C data. Red represents the interactions involved in PIRs containing TXNIP 3’UTR. Blue represents interactions containing TXNIP promoter. Height of each ribbon represents the average CHiCAGO score (Y-axis) of the corresponding interaction across 5 major blood cells including monocytes, neutrophils, CD4 + T-cells, CD8 + T-cells and B-cells. e , Annotations of the RefSeq genes in the depicted region. f , Pearson correlation of DNAme at 33 CpGs in TXNIP and its 25kb flanking region. The associations of DNAme with mean-DCCT HbA1c at each CpG are obtained by the same linear regression model (n=499) indicated in panel b and shown as a Manhattan plot in the upper panel. HbA1c-assoc CpGs (FDR < 15%) are labeled in red font with the most significant cg19693031 underlined. Pair-wise correlations of DNAme of these CpGs were analyzed by Pearson correlation with coefficients shown as heatmap. Blue/red represents negative/positive correlation, respectively. The majority of the CpGs depict positive correlations including all 9 HbA1c-assoc CpGs (in red), while 5 CpGs (in blue) showed little to negative correlation with other CpGs. g , Ribbon plot representing the association of DNAme at cg19693031with expression of its nearby genes (including TXNIP ) in monocytes. Multiple linear regression models adjusting for covariates based were applied to DNAme at cg19693031and expression of each nearby gene within 500 bp distance (n=1202 monocytes) to identify DNAme-assoc genes with nominal p < 0.05 (two-sided tests based on t-statistic). Blue indicates negative association at FDR < 0.05, while grey indicates associations with p < 0.05. h , In-vitro DNA hypomethylation at 3 CpGs in the 3’UTR of TXNIP (including cg19693031) induced by high glucose (HG) treatment of human primary bone marrow CD34 + cells. DNAme was measured by amplicon-seq. i , Upregulation of TXNIP expression induced by HG treatment of human primary bone marrow CD34 + cells. Cells were cultured in medium (see ) containing 25mmol/L glucose (control), or same medium with the addition of 20mmol/L glucose (45mmol/L total) for 72 hours (HG treatment). RT-PCRs were performed in triplicate, and the data shown represent means of triplicates from one experiment. j , Color codes of chromatin states in the heatmaps for 15 and 18 chromatin states shown in panel c.

Article Snippet: Human primary bone marrow CD34 + cells (1M-101C, Lonza, Switzerland,) were cultured in growth medium (#09605, STEMCELL Technologies, MA), STEMSPAN II plus StemSpanTM CD34+ Expansion Supplement (#02691, STEMCELL Technologies, MA) containing 25mmol/L glucose (control), or same medium with the addition of 20mmol/L glucose for 72 hours (HG treatment).

Techniques: Labeling, Expressing, In Vitro, Amplification, Cell Culture, Control